Natamycin

The standard serial dilution technique was used for the isolation of bacteria from soil samples collected from 7 different sites like the garden, the playground, near the canteen, near the sewage sump, near the biotechnology department, near RIT, and RUAPS. One gram of soil sample was mixed with l0 ml of sterile water and serially diluted (10⁻¹ to 10⁻⁴). From the serially diluted soil sample, 100 μl was mixed with warm nutrient agar medium and poured into Petri plates. Natamycin (Sigma-Aldrich, USA) at 20 μg/ml was amended with a molten nutrient agar medium at 50 C to prevent fungal growth [24 (link), 25 ]. After 48 h, the plates had a lawn of mixed bacterial colonies. The individual colonies were picked using sterile toothpicks and streaked onto fresh nutrient agar plates to get pure cultures. The pure culture was stored and used for testing antibacterial activity against human pathogenic bacteria Staphylococcus aureus (MTCC 96), Escherichia coli (MTCC 739), Pseudomonas aeruginosa (MTCC 741), and Klebsiella pneumoniae (MTCC 3040). The concentration of all pathogenic bacteria was adjusted to obtain the OD = 0.8 using a UV/Vis spectrophotometer at 600 nm.

Antifungal activity was evaluated against five strains isolated from the environment, food, or dairy products. They were chosen since they represent some of the most abundant fungal species causing significant food contamination [4 ]. The fungal strains were: Aspergillus niger (3071-13), Paecilomyces spp. (5332-9a), Mucor racemosus (LMA-722), Penicillium crustosum (27,159) and Rhodotorula mucilaginosa (27,173). The recovery method of the microorganisms from −80 °C, cell concentration count and the agar diffusion assay were described by Abou-Diab et al. [11 (link)]. As indicated by the Clinical and Laboratory Standards Institute (CLSI 2016, CLSI 2017), antifungal activity was measured as the diameter of the clear zone of growth inhibition and recorded as diameter of inhibition in millimeters. Natamycin (16.7 µg/mL) from Sigma-Aldrich (Oakville, ON, Canada) was used as positive control and sterile distilled water as negative control. All data are expressed as mean ± SD and are the mean of three replicates.

The Amphotericin B solution (product code L0009) was purchased from Biowest (VWR International GmbH, Darmstadt, Germany). Filipin III (from Streptomyces filipinensis, product code F4767), natamycin (product code 32417), and sodium deoxycholate (product code D6750) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Nystatin (product code 15340029) was purchased from Gibco (Thermo Fisher Scientific, Waltham, Massachusetts, United States). Ethanol (product code 9065.4) and mEthanol (CP43.3) were purchased from Carl Roth (GmbH + Co., KG, Karslruhe, Germany).

To ~50 g of berries, 47 ml of sterile distilled water and 3 ml of filtered absolute ethanol (99.8%; Sigma-Aldrich; Milan, Italy) were added. The suspension was mixed for 1 min, using the Seward Stomacher 400 (Seward Ltd, Technology Center, Worthing, West Sussex, UK) and incubated at room temperature for 7 days (Gullo et al., 2009 (link)). The obtained enrichment was inoculated on the surface of Glucose-Yeast-Extract-Calcium Carbonate Agar (GYC medium; glucose 5%, yeast extract 1%, CaCO3 1%, agar 1.5%, pH 6.8 ± 0.2; Scharlau Microbiology, Barcelona, Spain) supplemented with 100 mg/l of natamycin (Sigma-Aldrich, cod.: 32417) to inhibit fungal growth and incubated at 30°C for 5–10 days (Sengun and Karabiyikli, 2011 (link)). If no growth was noted, the enrichment broth was centrifuged, and inoculum from the sediment was streaked again on the GYC media plates. Where there was an overgrowth, a 1:10 dilution method up to five log reduction was applied. Tiny colonies with a clear halo were re-streaked onto WL nutrient agar (Oxoid, Ltd., Basingstoke, Hampshire, England), supplemented with cycloheximide (2 mg/l) (Sigma-Aldrich), and incubated at 25°C for 42–72 h. Colonies with a yellow halo were selected for further analysis and named with the prefix “G.”

High-purity commercial standards of SB, PS, and natamycin were purchased from Sigma-Aldrich (Wisconsin, USA). Other reagents of HPLC such as methanol, acetonitrile, ammonium acetate, glacial acetic acid, chloridric acid, and petroleum benzene were obtained from Merck (Darmstradt, Germany). Deionized water was prepared through the Millipore Milli-Q water purification system (ELGA, UHQ-II-MK3, and UK).

P. pluvialis isolates collected in the New Zealand regions of Auckland (n = 1), Bay of Plenty/Wanganui (n = 1), Coromandel (n = 2), Gisborne (n = 7), Hawke’s Bay (n = 1), Nelson (n = 3), Northland (n = 5), Taranaki (n = 1), Taupo (n = 1) and Wairarapa (n = 1) were obtained from the New Zealand Forest Research Institute (Scion) (Rotorua, New Zealand). Among these P. pluvialis, two are isolates whose cluster is unknown, 12 isolates belong to New Zealand cluster 1 (NZ1), and nine isolates belong to New Zealand cluster 2 (NZ2) [9 (link)]. Moreover, one isolate was isolated from Douglas-fir (Pseudotsuga menziesii), and 22 isolates were sourced from radiata pine (Pinus radiata) (Table S1). Isolates were maintained on V8 agar [26 ] supplemented with 250 mg/L ampicillin (AppliChem GmbH, Darmstadt, Germany), 10 mg/L natamycin (Sigma-Aldrich^®, St. Louis, MO, USA) and 250 mg/L rifampicin (Sigma-Aldrich^®, St. Louis, MO, USA) at room temperature in the dark.